Histone acetyltransferase 1 is a succinyltransferase for histones and non‐histones and promotes tumorigenesis

Lysine succinylation (Ksucc) is an evolutionarily conserved and widespread post‐translational modification. Histone acetyltransferase 1 (HAT1) is a type B histone acetyltransferase, regulating the acetylation of both histone and non‐histone proteins. However, the role of HAT1 in succinylation modulation remains unclear. Here, we employ a quantitative proteomics approach to study succinylation in HepG2 cancer cells and find that HAT1 modulates lysine succinylation on various proteins including histones and non‐histones. HAT1 succinylates histone H3 on K122, contributing to epigenetic regulation and gene expression in cancer cells. Moreover, HAT1 catalyzes the succinylation of PGAM1 on K99, resulting in its increased enzymatic activity and the stimulation of glycolytic flux in cancer cells. Clinically, HAT1 is significantly elevated in liver cancer, pancreatic cancer, and cholangiocarcinoma tissues. Functionally, HAT1 succinyltransferase activity and the succinylation of PGAM1 by HAT1 play critical roles in promoting tumor progression in vitro and in vivo. Thus, we conclude that HAT1 is a succinyltransferase for histones and non‐histones in tumorigenesis.     

Lysine succinylation and lysine malonylation in histones

Histone protein post-translational modifications (PTMs) are significant for gene expression and DNA repair. Here we report the identification and validation of a new type of PTM in histones, lysine succinylation. The identified lysine succinylated histone peptides were verified by MS/MS of synthetic peptides, HPLC co-elution, and isotopic labeling. We identified 13, 7, 10, and 7 histone lysine succinylation sites in HeLa, mouse embryonic fibroblast, Drosophila S2, and Saccharomyces cerevisiae cells, respectively. We demonstrated that this histone PTM is present in all eukaryotic cells we examined. Mutagenesis of succinylation sites followed by functional assays implied that histone lysine succinylation can cause unique functional consequences. We also identified one and two histone lysine malonylation sites in HeLa and S. cerevisiae cells, respectively. Our results therefore increase potential combinatorial diversity of histone PTMs and suggest possible new connections between histone biology and metabolism.     

SIRT5 desuccinylates and activates SOD1 to eliminate ROS

Cu/Zn superoxide dismutase (SOD1) is a key antioxidant enzyme. Deficiency of SOD1 is associated with various human diseases, including cancer. Here, we report that SOD1 is succinylated and that succinylation decreases its activity. SIRT5 binds to, desuccinylates and activates SOD1. SOD1-mediated ROS reduction is increased when SIRT5 is co-expressed. Furthermore, mutation of the SOD1 succinylation site inhibits the growth of lung tumor cells. These results reveal a novel post-translational regulation of SOD1 by means of succinylation and SIRT5-dependent desuccinylation, which is important for the growth of lung tumor cells.     

A Qualitative Proteome-Wide Lysine Succinylation Profiling of Tea Revealed its Involvement in Primary Metabolism

Lysine succinylation of proteins has potential impacts on protein structure and function, which occurs on post-translation level. However, the information about the succinylation of proteins in tea plants is limited. In the present study, the significant signal of succinylation in tea plants was found by western blot. Subsequently, we performed a qualitative analysis to globally identify the lysine succinylation of proteins using high accuracy nano LC-MS/MS combined with affinity purification. As a result, a total of 142 lysine succinylation sites were identified on 86 proteins in tea leaves. The identified succinylated proteins were involved in various biological processes and a large proportion of the succinylation sites were presented on proteins in the primary metabolism, including glyoxylate and dicarboxylate metabolism, TCA cycle and glycine, serine and threonine metabolism. Moreover, 10 new succinylation sites were detected on histones in tea leaves. The results suggest that succinylated proteins in tea plants might play critical regulatory roles in biological processes, especially in the primary metabolism. This study not only comprehensively analyzed the lysine succinylome in tea plants, but also provided valuable information for further investigating the functions of lysine succinylation in tea plants.     

In-depth analysis of the Sirtuin 5-regulated mouse brain malonylome and succinylome using library-free data-independent acquisitions

Post-translational modifications (PTMs) dynamically regulate proteins and biological pathways, typically through the combined effects of multiple PTMs. Lysine residues are targeted for various PTMs, including malonylation and succinylation. However, PTMs offer specific challenges to mass spectrometry-based proteomics during data acquisition and processing. Thus, novel and innovative workflows using data-independent acquisition (DIA) ensure confident PTM identification, precise site localization, and accurate and robust label-free quantification. In this study, we present a powerful approach that combines antibody-based enrichment with comprehensive DIA acquisitions and spectral library-free data processing using directDIA (Spectronaut). Identical DIA data can be used to generate spectral libraries and comprehensively identify and quantify PTMs, reducing the amount of enriched sample and acquisition time needed, while offering a fully automated workflow. We analyzed brains from wild-type and Sirtuin 5 (SIRT5)-knock-out mice, and discovered and quantified 466 malonylated and 2211 succinylated peptides. SIRT5 regulation remodeled the acylomes by targeting 164 malonylated and 578 succinylated sites. Affected pathways included carbohydrate and lipid metabolisms, synaptic vesicle cycle, and neurodegenerative diseases. We found 48 common SIRT5-regulated malonylation and succinylation sites, suggesting potential PTM crosstalk. This innovative and efficient workflow offers deeper insights into the mouse brain lysine malonylome and succinylome.     

Proteome-Wide Identification of Lysine Succinylation in the Proteins of Tomato (Solanum lycopersicum)

Post-translational modification of proteins through lysine succinylation plays important regulatory roles in living cells. Lysine succinylation was recently identified as a novel post-translational modification in Escherichia coli, yeast, Toxoplasma gondii, HeLa cells, and mouse liver. Interestingly, only a few sites of lysine succinylation have been detected in plants to date. In this study, we identified 347 sites of lysine succinylation in 202 proteins in tomato by using high-resolution mass spectrometry. Succinylated proteins are implicated in the regulation of diverse metabolic processes, including chloroplast and mitochondrial metabolism. Bioinformatic analysis showed that succinylated proteins are evolutionarily conserved and involved in various cellular functions such as metabolism and epigenetic regulation. Moreover, succinylated proteins exhibit diverse subcellular localizations. We also defined six types of definitively conserved succinylation motifs. These results provide the first in-depth analysis of the lysine succinylome and novel insights into the role of succinylation in tomato, thereby elucidating lysine succinylation in the context of cellular physiology and metabolite biosynthesis in plants.     

SuccSite: Incorporating Amino Acid Composition and Informative k-spaced Amino Acid Pairs to Identify Protein Succinylation Sites

Protein succinylation is a biochemical reaction in which a succinyl group (-CO-CH2-CH2-CO-) is attached to the lysine residue of a protein molecule. Lysine succinylation plays important regulatory roles in living cells. However, studies in this field are limited by the difficulty in experimentally identifying the substrate site specificity of lysine succinylation. To facilitate this process, several tools have been proposed for the computational identification of succinylated lysine sites. In this study, we developed an approach to investigate the substrate specificity of lysine succinylated sites based on amino acid composition. Using experimentally verified lysine succinylated sites collected from public resources, the significant differences in position-specific amino acid composition between succinylated and non-succinylated sites were represented using the Two Sample Logo program. These findings enabled the adoption of an effective machine learning method, support vector machine, to train a predictive model with not only the amino acid composition, but also the composition of k-spaced amino acid pairs. After the selection of the best model using a ten-fold cross-validation approach, the selected model significantly outperformed existing tools based on an independent dataset manually extracted from published research articles. Finally, the selected model was used to develop a web-based tool, SuccSite, to aid the study of protein succinylation. Two proteins were used as case studies on the website to demonstrate the effective prediction of succinylation sites. We will regularly update SuccSite by integrating more experimental datasets. SuccSite is freely accessible at http://csb.cse.yzu.edu.tw/SuccSite/.     

The yeast mitochondrial succinylome: Implications for regulation of mitochondrial nucleoids

Acylation modifications, such as the succinylation of lysine, are post-translational modifications and a powerful means of regulating protein activity. Some acylations occur nonenzymatically, driven by an increase in the concentration of acyl group donors. Lysine succinylation has a profound effect on the corresponding site within the protein, as it dramatically changes the charge of the residue. In eukaryotes, it predominantly affects mitochondrial proteins because the donor of succinate, succinyl-CoA, is primarily generated in the tricarboxylic acid cycle. Although numerous succinylated mitochondrial proteins have been identified in Saccharomyces cerevisiae, a more detailed characterization of the yeast mitochondrial succinylome is still lacking. Here, we performed a proteomic MS analysis of purified yeast mitochondria and detected 314 succinylated mitochondrial proteins with 1763 novel succinylation sites. The mitochondrial nucleoid, a complex of mitochondrial DNA and mitochondrial proteins, is one of the structures whose protein components are affected by succinylation. We found that Abf2p, the principal component of mitochondrial nucleoids responsible for compacting mitochondrial DNA in S. cerevisiae, can be succinylated in vivo on at least thirteen lysine residues. Abf2p succinylation in vitro inhibits its DNA-binding activity and reduces its sensitivity to digestion by the ATP-dependent ScLon protease. We conclude that changes in the metabolic state of a cell resulting in an increase in the concentration of tricarboxylic acid intermediates may affect mitochondrial functions.     

miR‐124 interacts with the Notch1 signalling pathway and has therapeutic potential against gastric cancer

Aberrant Notch signalling plays an important role in cancer progression. However, little is known about the interaction between miRNA and the Notch signalling pathway and its role in gastric cancer (GC). In this study, we found that miR‐124 was down‐regulated in GC compared with adjacent normal tissue. Forced expression of miR‐124 inhibited GC cell growth, migration and invasion, and induced cell cycle arrest. miR‐124 negatively regulated Notch1 signalling by targeting JAG1. miR‐124 levels were also shown to be inversely correlated with JAG1 expression in GC. Furthermore, we found that the overexpression of the intracellular domain of Notch1 repressed miR‐124 expression, promoted GC cell growth, migration and invasion. Conversely, blocking Notch1 using a γ‐secretase inhibitor up‐regulated miR‐124 expression, inhibited GC cell growth, migration and invasion. In conclusion, our data demonstrates a regulatory feedback loop between miR‐124 and Notch1 signalling in GC cells, suggesting that the miR‐124/Notch axis may be a potential therapeutic target against GC.     

Lysine succinylation of Mycobacterium tuberculosis isocitrate lyase (ICL) fine-tunes the microbial resistance to antibiotics

Lysine succinylation (Ksucc) is a newly identified protein posttranslational modification (PTM), which may play an important role in cellular physiology. However, the role of lysine succinylation in antibiotic resistance remains elusive. Isocitrate lyase (ICL) is crucial for broad-spectrum antibiotics tolerance in Mycobacterium tuberculosis (Mtb). We previously found that MtbICL (Rv0467) has at least three succinylated lysine residues, namely K189, K322, and K334.To explore the effect of succinylation on the activity of MtbICL, mutants’ mimicry of the lysine succinylation were generated by site-directed mutagenesis. ICL-K189E mutant strain is more sensitive than the wild-type to rifampicin and streptomycin, but not isoniazid. For the in vitro activity of the purified isocitrate lyase, only K189E mutant showed significantly decreased activity. Crystal structure analysis showed that Lys189 Glu dramatically increased the pKa of Glu188 and decreased the pKa of Lys190, whereas had negligible effect on other residues within 5?Å as well as disruption of the electrostatic interaction between Lys189 and Glu182, which might prevent the closure of the active site loop and cause severe reduction of the enzyme activity. Considering the genetic, biochemical, and crystallographical evidences together, the succinylation of specific ICL residue can fine-tune the bacterial resistance to selected antibiotics. The decreased enzymatic activity resulting from the succinylation-changed electrostatic interaction might underlie this phenotype. This study provided the first insight into the link between lysine succinylation and antibiotic resistance.     

Interplay between Trx‐1 and S100P promotes colorectal cancer cell epithelial–mesenchymal transition by up‐regulating S100A4 through AKT activation

We previously reported a novel positive feedback loop between thioredoxin‐1 (Trx‐1) and S100P, which promotes the invasion and metastasis of colorectal cancer (CRC). However, the underlying molecular mechanisms remain poorly understood. In this study, we examined the roles of Trx‐1 and S100P in CRC epithelial‐to‐mesenchymal transition (EMT) and their underlying mechanisms. We observed that knockdown of Trx‐1 or S100P in SW620 cells inhibited EMT, whereas overexpression of Trx‐1 or S100P in SW480 cells promoted EMT. Importantly, S100A4 and the phosphorylation of AKT were identified as potential downstream targets of Trx‐1 and S100P in CRC cells. Silencing S100A4 or inhibition of AKT phosphorylation eliminated S100P‐ or Trx‐1‐mediated CRC cell EMT, migration and invasion. Moreover, inhibition of AKT activity reversed S100P‐ or Trx‐1‐induced S100A4 expression. The expression of S100A4 was higher in human CRC tissues compared with their normal counterpart tissues and was significantly correlated with lymph node metastasis and poor survival. The overexpression of S100A4 protein was also positively correlated with S100P or Trx‐1 protein overexpression in our cohort of CRC tissues. In addition, overexpression of S100P reversed the Trx‐1 knockdown‐induced inhibition of S100A4 expression, EMT and migration and invasion in SW620 cells. The data suggest that interplay between Trx‐1 and S100P promoted CRC EMT as well as migration and invasion by up‐regulating S100A4 through AKT activation, thus providing further potential therapeutic targets for suppressing the EMT in metastatic CRC.     

IL‐17A promotes cell migration and invasion of glioblastoma cells via activation of PI3K/AKT signalling pathway

Glioblastomas (GBMs) are the most common of both benign and malignant primary brain tumours, in which the inflammatory and immunologic abnormalities are involved. Interleukin‐17A (IL‐17A) plays an important role in various inflammatory diseases and cancers. Several recent studies revealed that the expression of IL‐17A was overexpressed in human GBMs tissue. However, the accurate role of IL‐17A in GBMs remains unclear. In this study, we aimed to explore the effect of IL‐17A on cell migration and invasion of GBMs and the mechanism by which the effects occurred. We found that exogenous IL‐17A promoted significantly cell migration and invasion abilities in two GBMs cell lines (U87MG and U251) in a time‐dependent manner. In addition, the protein expressions of PI3K, Akt and MMP‐2/9 were increased in the GBMs cells challenged by IL‐17A. Furthermore, a tight junction protein ZO‐1 was down‐regulated but Twist and Bmi1 were up‐regulated. Treatment with a PI3K inhibitor (LY294002) significantly reduced the abilities of both migration and invasion in U87MG and U251 cells. LY294002 treatment also attenuated the IL‐17A causing increases of protein levels of PI3K, AKT, MMP‐2/9, Twist and the decreases of protein level of ZO‐1 in the U87MG and U251 cells. Taken together, we concluded that IL‐17A promotes the GBM cells migration and invasion via PI3K/AKT signalling pathway. IL‐17A and its related signalling pathways may be potential therapeutic targets for GBM.     

Lysine Succinylation of VBS Contributes to Sclerotia Development and Aflatoxin Biosynthesis in Aspergillus flavus

Aspergillus flavus is a common saprophytic and pathogenic fungus, and its secondary metabolic pathways are one of the most highly characterized owing to its aflatoxin (AF) metabolite affecting global economic crops and human health. Different natural environments can cause significant variations in AF synthesis. Succinylation was recently identified as one of the most critical regulatory post-translational modifications affecting metabolic pathways. It is primarily reported in human cells and bacteria with few studies on fungi. Proteomic quantification of lysine succinylation (Ksuc) exploring its potential involvement in secondary metabolism regulation (including AF production) has not been performed under natural conditions in A. flavus. In this study, a quantification method was performed based on tandem mass tag labeling and antibody-based affinity enrichment of succinylated peptides via high accuracy nano-liquid chromatography with tandem mass spectrometry to explore the succinylation mechanism affecting the pathogenicity of naturally isolated A. flavus strains with varying toxin production. Altogether, 1240 Ksuc sites in 768 proteins were identified with 1103 sites in 685 proteins quantified. Comparing succinylated protein levels between high and low AF-producing A. flavus strains, bioinformatics analysis indicated that most succinylated proteins located in the AF biosynthetic pathway were downregulated, which directly affected AF synthesis. Versicolorin B synthase is a key catalytic enzyme for heterochrome B synthesis during AF synthesis. Site-directed mutagenesis and biochemical studies revealed that versicolorin B synthase succinylation is an important regulatory mechanism affecting sclerotia development and AF biosynthesis in A. flavus. In summary, our quantitative study of the lysine succinylome in high/low AF-producing strains revealed the role of Ksuc in regulating AF biosynthesis. We revealed novel insights into the metabolism of AF biosynthesis using naturally isolated A. flavus strains and identified a rich source of metabolism-related enzymes regulated by succinylation.     

Helix stability in succinylated and acetylated ovalbumins: effect of high pH, urea and guanidine hydrochloride

Previous studies (Batra, P.P., Roebuck, M.A. and Uetrecht, D. (1990) J. Protein Chem. 9, 37-44) showed that succinylation or acetylation of 75% of the lysine residues has little effect on the secondary structure of ovalbumin. The acylation of the remaining 25% lysine residues, which apparently are partially buried, results in a substantial loss of the helical structure. These conformational changes may be due not only to electrostatic repulsions introduced by succinylation or acetylation of the positively charged epsilon-amino groups but also to steric hindrance, since an increase in the ionic strength failed to reverse the loss of the helical structure. An increase in pH to 12.2 results in a complete helix-to-coil transition in the maximally succinylated ovalbumin (but not in the partially succinylated or in any of the acetylated ovalbumins including the maximally acetylated derivative), perhaps because it is most expanded and its molecular interior most accessible to solvent as succinylation replaces +1 charge of epsilon-amino group with a -1 charge so that a net of -2 charge per succinyl group is placed on the protein molecule. This helix-to-coil transition in the maximally succinylated ovalbumin induced by high pH is fully reversed by increasing the ionic strength, indicating that only electrostatic effects are responsible for this disruption. Studies have also shown that although there is no loss of the helical structure until after the 75% surface lysine residues have been acylated, the helical structure does become progressively destabilized with increasing degree of modification, a conclusion drawn from urea unfolding curves. This destabilization of the helical structure is due primarily to electrostatic effects, as an increase in the ionic strength led to an increase in the urea transition mid-point. Unlike urea, the guanidine hydrochloride unfolding curves indicate that the transition mid-point for the native protein, as well as for the maximally succinylated and acetylated derivatives, is about the same, perhaps because the denaturant itself acts as an electrolyte.     

The role of loop and beta-turn residues as structural and functional determinants for the lipoyl domain from the Escherichia coli 2-oxoglutarate dehydrogenase complex

The lipoyl domain of the dihydrolipoyl succinyltransferase (E2o) component of the 2OGDH (2-oxoglutarate dehydrogenase) multienzyme complex houses the lipoic acid cofactor through covalent attachment to a specific lysine side chain residing at the tip of a beta-turn. Residues within the lipoyl-lysine beta-turn and a nearby prominent loop have been implicated as determinants of lipoyl domain structure and function. Protein engineering of the Escherichia coli E2o lipoyl domain (E2olip) revealed that removal of residues from the loop caused a major structural change in the protein, which rendered the domain incapable of reductive succinylation by 2-oxoglutarate decarboxylase (E1o) and reduced the lipoylation efficiency. Insertion of a new loop corresponding to that of the E. coli pyruvate dehydrogenase lipoyl domain (E2plip) restored lipoylation efficiency and the capacity to undergo reductive succinylation returned, albeit at a lower rate. Exchange of the E2olip loop sequence significantly improved the ability of the domain to be reductively acetylated by pyruvate decarboxylase (E1p), retaining approx. 10-fold more acetyl groups after 25 min than wild-type E2olip. Exchange of the beta-turn residue on the N-terminal side of the E2o lipoyl-lysine DK(A)/(V) motif to the equivalent residue in E2plip (T42G), both singly and in conjunction with the loop exchange, reduced the ability of the domain to be reductively succinylated, but led to an increased capacity to be reductively acetylated by the non-cognate E1p. The T42G mutation also slightly enhanced the lipoylation rate of the domain. The surface loop is important to the structural integrity of the protein and together with Thr42 plays an important role in specifying the interaction of the lipoyl domain with its partner E1o in the E. coli 2OGDH complex.     

Understanding the role of internal lysine residues of serum albumins in conformational stability and bilirubin binding

The role of internal lysine residues of different serum albumins, viz. from human, rabbit, goat, sheep and buffalo (HSA, RbSA, GSA, SSA and BuSA), in conformational stability and bilirubin binding was investigated after blocking them using acetylation, succinylation and guanidination reactions. No significant change in the secondary structure was noticed whereas the tertiary structure of these proteins was slightly altered upon acetylation or succinylation as revealed by circular dichroism (CD), fluorescence and gel filtration results. Guanidination did not affect the native protein conformation to a measurable extent. Scatchard analysis, CD and absorption spectroscopic results showed marked reductions (5-21-fold decrease in K(a) and approximately 50% decrease in the CD Cotton effect intensity) in the affinity of albumins for bilirubin upon acetylation or succinylation whereas guanidination produced a small change. Interestingly, monosignate CD spectra of bilirubin complexed with GSA, SSA and BuSA were transformed to bisignate CD spectra upon acetylation or succinylation of internal lysine residues whereas spectra remained bisignate in the case of bilirubin bound to acetylated or succinylated derivatives of HSA and RbSA. When probed by CD spectroscopy, bilirubin bound to acetylated or succinylated derivatives of GSA and SSA rapidly switched over to native albumins and not vice versa. These results suggested that salt linkage(s) contributed by internal lysine residue(s) play an important role in the high-affinity binding of bilirubin to albumin and provide stability to the native three-dimensional conformation of the bound pigment. Chloroform severely decreased the intensity of both positive and negative CD Cotton effects of bilirubin complexed with acetylated or succinylated derivatives of all albumins which otherwise increased significantly in the case of bilirubin complexed with native and guanidinated albumin derivatives, except the bilirubin-RbSA complex which showed a small decrease in intensity. These results suggest that the presence of salt linkage(s) in bilirubin-albumin complexation is(are) crucial to bring about effective and efficient stereochemical changes in the bound pigment by co-binding of chloroform which seems to have at least one conserved binding site on these albumins that is shared with bilirubin.     

Influence of succinylation of bovine serum albumin on its conformation and bilirubin binding

In order to investigate the role of lysine residues in the interaction of bilirubin with bovine serum albumin, five succinylated preparations of albumin, namely: 23%, 39%, 49%, 55% and 87%, were prepared, and their conformational and bilirubin-binding properties were studied by the techniques of gel filtration, ultraviolet and visible spectroscopy, and fluorescence quenching. Gel filtration experiments performed at pH 7.0 and ionic strengths 0.15 and 1.0 suggested that the albumin molecule undergoes gradual disorganization with increase in succinylation. The Stokes radius and frictional ratio at ionic strength 0.15 increased from 3.7 nm and 1.36, respectively, for the native protein to 6.3 nm and 2.26 for maximally (87%) succinylated albumin. Interestingly, increase in ionic strength to 1.0 caused significant refolding in succinylated preparations as evidenced by a decrease in Stokes radius and frictional ratio (5.3 nm and 1.90 for 87% succinylated albumin). Progressive succinylation produced a steady decline in the intensity of bilirubin-induced fluorescence quenching, and in the visible spectral changes of the bilirubin-albumin complex at 480 nm. Both of these changes had a good correlation with increase in Stokes radius. Increase in ionic strength to 1.0 produced a significant reversal in these properties. From these results we conclude that probably none of the surface lysine residues is involved in bilirubin-albumin interaction, and that if lysine residues are involved in this interaction they must be buried in the protein interior.